Protein kinase C (PKC) isoforms are involved in diverse signalling pathways, and aberrant activity of PKCs has been implicated in pathological states, including cancer, autoimmunity, ischaemia and diabetes [1]. In contrast with the highly-conserved ATP- and substrate-binding sites in the C-terminal kinase domain, the N-terminal C2 domain in some PKC isoforms interacts with distinct regulatory proteins, thus providing a potential means for isoform-specific targeting by small molecules.
PKCε is associated with insulin resistance in type II diabetes [2]. Upon activation of PKCε, the PKCε C2 domain is bound by the scaffolding protein Receptor for Activated C-Kinase 2 (RACK2), leading to translocation of PKCε to the cell membrane and inhibition of insulin receptor kinase activity. A putative RACK2-binding site in PKCε C2, designated εV1-2, has been identified as an antagonist of PKCε translocation [3]. It has also been observed that a site within PKCε C2, pseudoεRACK (ψεRACK), bears some similarity to a sequence within RACK2. A ψεRACK peptide has been shown to enhance PKCε translocation in vivo, leading to the proposition that an intramolecular interaction between the ψεRACK and εV1-2 sites stabilises the inactive form of PKCε [4]. Characterisation of these regulatory interactions would facilitate development of PKCε-selective inhibitors.
In the present work, direct interactions between PKCε, RACK2 and εV1-2/ψεRACK peptides are tested by SPR and NMR. The binding of full-length RACK2 to the εV1-2 peptide and inhibition of the PKCε-RACK2 interaction by εV1-2 are confirmed. We further demonstrate that the PKCε C2 domain participates in an intramolecular interaction with full-length PKCε, but none of the εV1-2, ψεRACK or RACK2-derived peptides are able to bind PKCε. These results support the involvement of the εV1-2 site in the PKCε-RACK2 interaction, and indicate that further work is required to determine the PKCε-binding site of RACK2 and the site(s) involved in PKCε intramolecular interactions.